Limb trauma can lead to the development of a complex regional pain syndrome (CRPS), a potentially debilitating chronic pain state. Currently there is no consensus on either the pathophysiology or treatment for CRPS and translational studies are clearly needed to identify therapeutic targets and effective treatments. We have developed a tibia fracture rodent model that closely replicates the CRPS clinical scenario with hindlimb unweighting, allodynia, warmth, edema, bone loss, exaggerated neuroinflammatory responses and increased levels of inflammatory mediators (TNF, IL-1, and NGF) expressed by keratinocytes in the affected skin. This proposal tests the hypothesis that fracture and immobilization can enhance sensory and sympathetic efferent signaling in skin keratinocytes and dorsal root ganglion (DRG) glial cells, causing cellular proliferation and inflammatory mediator release leading to the development of chronic inflammation and pain. The primary objective of this proposal is to identify target-specific treatments for CRPS using approved or soon-to-be-approved medications. The specific aims are: 1) to identify the pro-inflammatory effects of sensory neuropeptides and adrenergic agonists in cultured keratinocyte, Schwann, and DRG cells, 2) to determine whether sensory and sympathetic efferent signaling is up-regulated after fracture and three- week cast immobilization in mice and to establish the role this neuronal signaling plays in the development of post-fracture CRPS-like changes in the hind limb and inflammatory mediator expression in the skin and DRGs innervating the injured limb, and 3) to characterize the effects of immobilization and remobilization on post-fracture CRPS-like changes, sensory and sympathetic signaling, and inflammatory mediator expression. In vitro cell culture studies in keratinocyte, Schwann, and DRG cell lines will be utilized to identify candidate neurotransmitters and receptor subtypes for translational mouse fracture studies. The fracture CRPS model will be utilized in mouse strains deficient for specific neurotransmitters or their receptors to determine the role played by sensory and sympathetic neurotransmitter signaling in the development of fracture induced pain, inflammation, and bone loss. Additional studies will determine the effects of 1) cast immobilization in intact mice, 2) early remobilization in fractured and intramedullary pinned mice, and 3) daily treadmill exercise therapy in fracture and casted mice, looking at the effects of these interventions on the development of pain behaviors, edema, warmth, and inflammatory mediator expression in the skin keratinocytes and the DRG satellite glia. We anticipate that these experiments will help identify the neural systems, transmitters, and peripheral cellular targets that modulate post-fracture inflammation and pain, discover novel anti- inflammatory effects for mobilization and exercise after trauma, and generate translational data identifying specific molecular targets for future CRPS trials. PUBLIC HEALTH RELEVANCE: We anticipate that the proposed studies in this application will provide a sound experimental foundation supporting the neuroinflammatory basis of complex regional pain syndrome (CRPS). Identifying the relevant inflammatory signaling pathways leading to the development of post-traumatic CRPS could readily translate into more effective CRPS drug treatments. If funded, this work would be an important step towards the ultimate goal of improving the clinical management of this debilitating condition.